Gene targeting in large animals has the potential to be useful in agriculture. In this study, we examined whether an �1,3-galactosyltransferase-gene knockout (null) calf could be produced using a single targeting vector for disruption of both alleles and a rejuvenation of cell line by production of cloned fetuses. A promoter-less positive selection vector (pGT-22) containing an IRES (internal ribosome entry site)-antibiotic resistance gene(neo) cassette and loxP sequences was used to disrupt the bovine �1,3-galactosyltransferase (�1,3-GT) gene. In gene-targeting (GT), Japanese Black fetal fibroblasts were transfected with pGT-22, and were selected with 0.4 mg mL-1 G418. G418-resistant cells were monitored by PCR and Southern blot analysis. After PCR selection, a portion of the PCR-positive colonies were infected with the adenovirus AxCANCre, which transiently expresses Cre recombinase in the infected cells, to excise the antibiotic-resistance gene cassette (IRES/lacZ-neo) from the targeted allele. The targeted cells in which homologous recombination occurred were used for somatic cell nuclear transfer (SCNT). The cell cycle synchronization of donor cells in the early G1 phase and SCNT were performed according to the established procedure in our laboratory (Urakawa et al. 2004 Theriogenology 62, 714–728). As a result, in the first GT, one PCR-Southern blot-positive clone (0.26%, in 380 G418-resistant colony) was used for SCNT. Of 35 pulsed SCNT embryos, 4 (11.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT+/-) was established and was transfected with pGT-22 in the second GT. As a result, 8 PCR-Southern blot-positive clones (1.6%, in 508 G418-resistant colony) were recovered. The 2nd GT and the following SCNT were accomplished by the same methods used in the 1st GT and SCNT procedure. Of 36 pulsed SCNT embryos, 7 (19.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT-/-) was established, and was used for SCNT to obtain the �1,3-GT homozygous knockout offspring. Finally, 52 of 148 pulsed SCNT embryos (35.1%) developed to the blastocyst stage and 6 cloned embryos were transferred into 3 recipient heifers. A single knockout female offspring with normal morphology in all organs was born (Day 287) but died about one hour after birth. In this study, we established both heterozygous and homozygous �1,3-GT knockout cell lines from primary fetal fibroblasts and produced an �1,3-GT knockout offspring using a single targeting vector and rejuvenated cell lines.
Screening of HaCaT Clones for CCL20 Gene Knockout and Preliminary Exploration of Gene-Targeting Vector Transfection Approaches in this Cell Line